Abstract:
Introduction. Chondral injuries are common following a knee trauma. There are numerous
studies with different ways to obtain a suitable graft for articular cartilage regeneration, but
without imposing results.
Material and methods. From two freshly sacrificed rabbits the distal femurs were harvested and
frozen at -84°C for one week. From each distal femur all tissues except cartilage and subcondral
bone were removed and small pieces of normal osteochondral tissue (NOCT) were taken. The
remaining osteochondral tissue has been demineralized in 0,6M HCl (Chem-Lab, Belgium) over
night and again small pieces of demineralized osteochondral tissue (OCDT) were cutted with a
scalpel and placed in a PBS solution for 24 hours. The remaining OCDT were separated in 4
groups. Two groups were decellularized in 0,5% and 1% SDS (Sigma, UK) and another two in
0,5% and 1% Triton X-100 (HiMedia, India). The decellularization lasted for 24 hours. At the
next day the decellularized and demineralized osteochondral tissues (OCDDT) were washed with
distilled water and PBS for 24 hours. All tissues were dessicated through centrifugation at 4000
rpm for 10 min (Hettich, Germany). From all types of OCT were cutted from three to nine pieces
20 mg each and quantification of DNA was performed with GeneJET Genomic DNA
Purification Kit (Thermo Fisher, Lithuania). The results were read with spectrophotometer
NanoDrop 2000c at wavelength of 260 nm (Thermo Fisher, USA). The best decellularized tissue
and OCDT were tested for cytotoxicity with MTT test (ISO 10993-5) with mesenchymal stem
cells and chondrocytes.
Results. The average of DNA content in a rabbit NOCT is 36 ng/μl, in OCDT 4,23 ng/μl,
OCDDT with 0,5% and 1% SDS is 3,23 ng/μl and 2,16 ng/μl respectively and in OCDDT with
0,5% and 1% TritonX-100 is 1,96 ng/μl and 0,96 ng/μl. At the MTT assay with mesenchymal
stem cells and chondrocytes on the OCDT and OCDDT with 1% TritonX-100, we obtained a
higher cell viability in both cases more than 80%.
Conclusions. Obtaining a suitable osteochondral tissue for cartilaginous tissue engineering is
very difficult because this process involves utilisation of a very toxic chemicals that harm this
tissue. A shorter exposure period to chemical agents and preliminary modeling of the graft is
mandatory. Also the OCDDT with 1% TritonX-100 shows the best results compared to others.
Description:
Laboratory of Tissue Engineering and Cells Cultures, Laboratory of genetics,
Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of Moldova
Department of Biomedical Sciences. Faculty of Medical Bioengineering “Grigore T.Popa"
University of Medicine and Pharmacy, Iasi, Romania